Sphingosine 1-phosphate (S1P) chemotactic gradients are necessary for correct temporal and spatial positioning of immune cells while the blood-tissue S1P gradient supports endothelial barrier integrity. We propose now to provide a new method of modulating the immune system by changing the lymph-lymph node S1P gradient, which is crucial for lymphocyte trafficking. The initial indication that S1P gradients have a pivotal role in immune cell migration was the discovery of the mechanism of action of the immunosuppressive drug, fingolimod. This drug desensitizes lymphocyte S1P1 receptors, which renders these cells unable to migrate from secondary lymphoid tissues to the S1P rich environment of efferent lymph. Although ultimately successful as a medicine, fingolimod and other S1P1 receptor agonists have on-target cardiac and vascular toxicities, which necessitates additional strategies to modulate the immune response by manipulating S1P signaling. Studies with mutant mice predict that a viable alternative strategy is to eliminate the lymph-lymph node S1P gradient by inhibiting the transporter, Spns2, which supplies S1P from endothelial cells to efferent lymph. However, the Spns2 inhibitors that are required to test this idea are not available. As a first step in meeting this need, we took advantage of the toxicity of high levels of S1P in Saccharomyces cerevisiae to build an S1P transporter assay. We used this assay to screen our focused chemical library of S1P agonists and sphingosine kinase inhibitors and identified a hit compound that, after minimal chemical manipulation, resulted in a lead compound that drives the lymphopenia and reduction in plasma S1P expected of an Spns2 inhibitor. Through iterative chemical synthesis and pharmacologic testing, we will optimize our lead Spns2 inhibitor as well as discover and optimize additional chemical series of Spns2 inhibitors. Ultimately, we will generate potent Spns2 inhibitors with the pharmacokinetic properties suitable for in vivo applications. The selectivity of the Spns2 inhibitors will be ascertained by rigorous counter-screening against other S1P interacting proteins including S1P receptors, catabolic and anabolic enzymes and another, erythrocyte-specific, S1P transporter, Mfsd2b. In developing Spns2 inhibitors, we will use plasma S1P levels and peripheral blood lymphocyte counts as biomarkers of target engagement. Spns2 inhibitors will be deployed in a battery of disease models where immune-modulation is indicated. Further, we will assess our Spns2 inhibitors for potential adverse events associated with S1P signaling including vascular leak, bradycardia and ototoxicity. Optimally, our studies will validate Spns2 as a therapeutic target for immune system modulation. At a minimum, we will provide reliable chemical tool for exploring the complex biology of S1P.